The purpose of this step is to remove any residual primers and reagents from the pooled Amplified Indexed Libraries so that the preparations are ready for NGS.
- Add 1.8x volume of Agencourt® AMPure® XP Reagent (at room temperature) to pooled Amplified Indexed Libraries and Negative Control sample mix in an Eppendorf tube, and pipet up and down 5 times to thoroughly mix the bead suspension with the pooled Amplified Indexed Libraries.
- Incubate the mixture for 2 minutes at room temperature.
- Place tube in the Thermo Fisher Dynabeads® MPC®-S Magnetic Stand for 1 minute or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet.
- Add 500 μl of freshly prepared 80% ethanol to the tube, and wash the beads by pipetting.
- Place the tube in the Magnetic Stand for 1 minute or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet.
- Repeat steps 4 and 5 for a second wash.
- Briefly centrifuge tubes at low speed and place the tubes in the Magnetic Stand. Use a 20-μl pipette to remove the residual ethanol droplets from the tube, and air-dry the beads at room temperature for 2 minutes.
- Add 25 μl of fresh PCR-Grade Water to the pellet to disperse the beads, and let stand for 1 minute.
- Place the tube on the Magnetic Stand for 1 minute. Transfer 20 μl of the supernatant to a new Eppendorf tube.
- Measure the concentration of both pooled Amplified Indexed Libraries sample and Negative Control sample using the Qubit® dsDNA High Sensitivity Assay. Subtract the concentration reading (ng/μl) of the Negative Control sample from the pooled Amplified Indexed Libraries reading.
- Dilute the pooled Amplified Indexed Libraries probe sample to 1.8 ng/μl, which corresponds to a concentration of 10nM for the following NGS step.
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